Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

Abstract

We show that in solutions of human hemoglobin (Hb)-oxy-and deoxy-Hb A or S-of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40°C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.T he primary pathogenic event in sickle cell anemia (1) is the polymerization of the mutated hemoglobin (Hb) S into linear fibers (2), mostly in the postcapillary venules (3-5), forming spherulitic domains, sheaves of parallel polymers (6-8), and other secondary structures, which stretch the erythrocytes and increase the intracellular viscosity leading to vasoclussion (9). In the absence of a curative treatment (2, 10), the main impetus for research has been on slowing and preventing the polymerization of deoxy-HbS (11). Recent experiments (12), simulations (13), and theory (14) suggest that the kinetics of formation of protein solid phases can be controlled by shifting the phase boundary for liquid-liquid (L-L) separation, occurring with some proteins (15)(16)(17). In this article, we discuss the tests of the first prerequisite for the action of this control mechanism-the existence of a dense liquid phase in the solutions of normal and sickle cell Hb at near-physiological conditions. Methods Procedures for Direct Monitoring of L-L Separation and Nucleation ofPolymers. Samples of Hb solutions (A or S) with concentration in the range 9.6-35 g͞dl, in oxygenated or deoxygenated state, in 0.15 M potassium phosphate buffer at pH 7.35, with 0.1-1% (wt͞vol) polyethylene glycol (PEG) of molecular mass 8,000 g͞mol (PEG 8000) were held between two microscope slides. The solution layer thickness, determined by focusing on imperfections on the bottom and top inside glass surfaces, was 10-40 m and the sample volume was a few microliters. The slides were sealed with Maunt-Quick (Daido Sangyo, Japan) sealant, and mounted on a custom-made temperature control stage. The latter consists of an aluminum block attached to thermoelectric (Peltier, Sterling, MA) coolers and has an opening that allows the approach of the microscope condenser from the slide bottom for differential interference contrast (DIC) imaging. The controller ensures temperature stability and control within Ϯ0.05°C between Ϫ5 and 70°C. For the detection of the HbS polymers, polymer bundle domains, and gels, we used a DIC-equipped microscope (Leitz Orthoplan, magnification ϫ1,000) as in refs. 6, 7, and 18. To avoid heat loss through the microscope lens or the condenser, they were both inserted in brass coolers through which we flowed water coming from a temperature-contr...

Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics

“…After formation of these denseliquid droplets, relaxation to the lower energy-ordered state occurs. Experimental studies of bulk protein crystallization (10,11) have led to similar conclusions and provide convincing evidence for a dense-liquid phase that precedes order. But due to experimental limitations associated with viewing assembly in three dimensions with molecular resolution, a molecular-scale picture of multistage pathways has not been obtained.…”

Effects of patch size and number within a simple model of patchy colloids

“…[54] This is clearly unrealistic for the majority of proteins where the distribution of hydrophobic surface groups is significantly irregular, a feature that can be captured, at the simplest level of description, by the model studied here. The specific location of the coexistence lines, such as those considered in the present study, have important consequences in the study of pathogenic events for sickle cell anemia [55] and other human diseases. The expansion in spherical harmonics Y lm (ω) of g (12) in an arbitrary space frame reads [48] g (12) = 4π…”

Crystallization by particle attachment in synthetic, biogenic, and geologic environments

“…1 and 4). Precursors can include one or more solid amorphous phases (10,12,14,15,18,30,46,64,65), dense liquids or gels (21,49,53), or crystalline nanoparticles (30,57,60,63). Each results in a distinct growth history, but whether or not the final outcomes are also distinct should depend on the extent to which monomer-by-monomer addition competes with the particle-attachment pathways and coarsening or recrystallization processes modify the structure and morphology of the growing crystal.…”

“…Protein and polymer solutions often exhibit partial miscibility, with a dense liquid phase (49,78) that can act as a precursor to crystal formation. The emergence of such a state, however, does not necessarily imply its active participation in crystallization.…”